Staphylococcus aureus is a major human pathogen that frequently causes pneumonia in normal infants and children, in patients with cystic fibrosis, and especially nosocomial infections in intensive care units. Staphylococcal infections in the lung are characterized by a polymorphonuclear leukocyte response. The organisms express many virulence factors that directly interact with components of the immunesystem, including immunoglobulin, T and B cells as well as platelets. In this proposal we will study in detail exactly how protein A, a major surface component of S.aureus, contributes to the pathogenesis of pneumonia by binding specifically to TNFR1 and activating TNFa signaling cascades in airway epithelial cells. An anti- inflammatory response is also initiated which includes the expression of the TNFa converting enzyme and the shedding of TNFR1 and IL-6 receptors. The binding of protein A and the effects of polymorphisms in spa linked to clinical outbreaks will be characterized by examining binding to TNFR1 and their relative potency in activating pro or anti-inflammatory signaling. A spa-GFP fusion will be used to document when protein A expression is activated in the murine lung. Whether protein A induces epithelial apoptosis via the TNF pathway will also be studied. Experiments to define how protein A activates a regulatory - anti-inflammatory cascade at the same time it stimulates TNF signaling are described. The relative importance of epithelial - TNFR1 activation by protein A, as opposedto signaling mediated by immune cells, will be defined in transgenic mice that express TNFR1 only in the airway epithelium. Since the control of inflammation is especially important in the airways, we will establish exactly how protein A and S.aureus induce TNFR1 and IL-6 receptor shedding through the activation of the TNFa converting enzyme. These studies should provide new insights into the pathogenesis of Staphylococcalpneumonia and suggest new targets for therapy. These studies will provide knowledge about the pathogenesis of Staphylococcal pneumonia, a major cause of infection in infants, immunocompromised hosts, patients with cystic fibrosis, and a major source of morbidity in intensive care units.